Regulated vesicle exocytosis is a Ca2+dependent membrane fusion event of key physiological importance for integration of the nervous, endocrine and immune systems. Because of its central role and involvement in disease, it is important to identify the proteins and mechanisms that are utilized for Ca2+regulated vesicle exocytosis. In neural & endocrine cells, the SNARE proteins VAMP-2, Syntaxin-1 & SNAP-25 constitute the core fusion machinery. SNAREs execute fusion by forming trans complexes between docked vesicles and the plasma membrane. However, not all docked vesicles are fusion-competent and a priming step is needed to promote a fusion-ready state. The assembly of SNARE complexes is fundamental to priming but poorly understood. The principal goal of our proposed research is to elucidate vesicle priming reactions. To do so, we will determine the mechanism of proteins that catalyze priming. Genetic studies indicate that Munc-13 and CAPS, which have sequence homology in a MUN domain, mediate vesicle priming possibly in concert with Munc-18. We found that Munc-13 exhibits a novel interaction with Syntaxin by binding the SNARE motif- containing H3 domain, which is different from the previously-suggested N-terminal Syntaxin binding. Further studies of this interaction and its role will likely shift the current paradigm for a molecular model of priming. We will exploit our findings that purified Munc-13 proteins reconstitute priming in permeabilized cells, promote SNARE-dependent liposome fusion, and stimulate SNARE complex formation in vitro to elucidate a new mechanism for Munc-13 function. This mechanism will be critically evaluated in neural, neuroendocrine and mast cells. The specific aims for this project are 1) To fully characterize Munc-13-Syntaxin interactions and generate mutant proteins deficient in binding; 2.) To determine the mechanism by which Munc-13 promotes SNARE complex assembly in vitro; 3) To critically assess the functional role of the new Munc-13 mechanism for priming in PC12 neuroendocrine, hippocampal neuronal, and RBL-2H3 mast cells; and 4) To determine how multiple MUN domain proteins (Munc-13 & CAPS) contribute to priming in secretory cells. Completion of the proposed work will provide major new insight into vesicle priming, Munc-13 mechanisms, and the fusion machinery for regulated vesicle exocytosis. PUBLIC HEALTH RELEVANCE: Neurotransmitters, peptide hormones, and inflammatory mediators are secreted from neural, endocrine and mast cells by the regulated fusion of vesicles with the plasma membrane. We will study key protein interactions that are needed to convert vesicles into a fusion-ready state. Because mutations in these proteins result in human disease (e.g., in immune system function), the work will contribute to understanding the underlying basis of these pathologies.